The term "Monoclonal Antibodies" refers generally to a substantially homogeneous immunoglobulin population of a defined specificity that is produced by a cloned cell line. Although immunologists had isolated many plasmacytomas producing such immunoglobulins, it was not until the seminal work of Kohler and Milstein that the construction of cell lines secreting homogeneous antibodies of predetermined specificity against an antigen became feasible (Kohler, G. and Milstein, C., Nature 256: 495-497 [1975]).
Briefly, Kohler and Milstein's original experiments utilized Sendai virus to fuse a HAT (hypoxanthine, aminopterin and thymidine)-sensitive variant of a mouse myeloma cell line and spleen cells from mice immunized against sheep red blood cells. When cultured on HAT medium for about one week, only hybrid cells (fusions of the myeloma and spleen cells) survived--the myeloma cells died because of their HAT sensitivity, while the remaining normal spleen cells generally are unable to survive in culture. These hybrid cells, known as hybridomas, contained the HGPRT (hypoxanthine guanine phosphoribosyl transferase) salvage mechanism from normal cells needed to survive in HAT medium, yet retained the immortality capability of the myeloma fusion partner. Moreover, when separated out into individual cell clones, it was discovered that some of the hybridomas secreted large quantities of homogeneous antibodies specific for sheep red blood cells. Thus, a simple and effective method of producing monoclonal antibodies was realized.
Later, the easier and safer polyethylene glycol fusion treatment was substituted for the viral fusion (Ringerty, N. and Savage, R. Cell Hybrids, Academic Press, New York [1976]) and now it is the primary fusion mechanism utilized (Galfre, G. and Milstein, C. Meth. Enzym. 73: 3-46 [1981]). Otherwise, the Kohler and Milstein technique remains essentially unchanged.
The technique found rapid and universal acceptance in the scientific community, and a variety of hybridomas producing monoclonal antibodies of various specificities have been produced. In fact, the strategies for hybridoma production,--including choice of antigen, fusion partner cell and subsequent cultivation--are legion. (See generally, Golding, J., Monoclonal Antibodies: Principles and Practice, Academic Press, New York [1983]). However, the production of a hybridoma capable of secreting monoclonal antibodies having a precise range of binding properties (e.g., specific for a mammalian protein but not for the same protein from other species--although the protein may be very similar structurally between species) remains a very difficult problem, and one often resolved only fortuitously.
Monoclonal antibodies find utility in a vast number of ways, including: immunoassays, immunohistochemical staining, immunoabsorbent, and cell sorting procedures. Moreover, when specific for a protein (or some other molecule having a measurable activity on cells), the monoclonal antibody can be used to ascertain the molecule's function by determining the effect of antibody-mediated removal on the cellular system under experimentation.
With the advent of recombinant DNA technology, monoclonal antibodies have added utility as aids in immunochemical blotting--the so-called "Western Blots". The antibodies are used to detect the production of exogenous proteins, such as hormones on electrophoresed gels.
One hormone that has attracted considerable attention recently is interleukin-2 (IL-2), originally known as T cell growth factor. This lymphokine was discovered just over 10 years ago (Smith, K. Immunol. Rev. 51: 337-357 [1980]), and its prime function is almost certainly the stimulation and maintenance of proliferation of T cells--cells crucial in the mammalian immune response. In fact, the removal of IL-2 from proliferating T cells results in their death within a few hours (Ruscetti; F. et al., J. Immunol. 123: 2928-2931 [1977]). Thus, some immunologists believe that IL-2 may be at the center of the entire immune response. For a detailed review of IL-2 activities, see Farrar, J. et al., Immunol. Rev. 63: 129-166 [1982], which is incorporated herein by reference.
Not surprisingly, researchers have applied monoclonal antibody technology in an attempt to develop a better understanding of IL-2's role in the imune response, such as by developing better methods for its assay and purification. By way of example, at least three European Patent Application (Nos. 82302231.4, 83108444.7, and 83112532.3) have been published concerning monoclonal antibodies capable of binding IL-2, and at least one commercial source also exists (DMS-1; Genzyme Corporation, Boston, MA).
Each of these monoclonal antibodies reportedly exhibits significant binding affinity for human IL-2, and some also exhibit cross-reactivity with mouse and/or rat IL-2. While affinity for human IL-2 is very important for many purposes, it would be useful to have a monoclonal antibody that recognizes an epitope (an antigenic site) on mouse IL-2 that does not exist on human or rat IL-2. This would permit the determination (serologically or otherwise) of mouse IL-2 in the presence of human IL-2. Also, purification of mouse IL-2 and two site immunoassays become more feasible. Moreover, this would allow the experimental suppression of endogenous IL-2 (e.g., mouse), while adding an endogenous source (e.g., human or rat)--expanding immunologists' knowledge base concerning IL-2's role in the mammalian immune response.
Thus, there exists a need for a hybridoma capable of producing a monoclonal antibody specific for mouse IL-2, but which does not significantly cross-react with human or rat IL-2. The present invention fulfills this need.